The goal of this project is to understand how newly synthesized proteins which must be secreted or inserted into membranes are identified and directed to transport sites in the endoplasmic reticulum (ER) or the cytoplasmic membrane in prokaryotic cells. The project combines biochemical and genetic approaches to investigate the role of the signal recognition particle (SRP), the signal recognition particle receptor (SRP receptor), and their bacterial homologs in this process. Previous studies have shown that the SRP ribonucleoprotein (RNP) recognizes nascent polypeptide chains bearing "signal sequences", which earmark proteins for entry into the ER, and then catalyzes their translocation across the membrane upon interaction with the ER-bound SRP receptor. A principal aim of this project is to elucidate the mechanism by which SRP recognizes signal sequences and then releases them in a regulated manner. By studying this problem we also expect to obtain insight into questions of broad biological significance, such as the regulation of multi-step pathways, the structural features which enable a protein to possess broad substrate specificity, and the functional roles of RNA. Previous work indicated that signal sequences bind to a site in the C- terminal domain of the 54kd subunit of mammalian SRP (SRP54). Two new lines of evidence, however, suggest that the N-terminal GTPase domain of the protein also plays a significant role in signal sequence recognition. Point mutations introduced into a conserved motif in this domain do not affect GTPase function, but rather reduce signal sequence binding activity. In addition, mutants of the E. coli homolog of SRP54 ("Ffh") which were selected on the basis of having moderately reduced signal sequence binding activity contain mutations primarily in the N-terminal domain. Because of methodological limitations to studying the SRP pathway in mammalian cells, we have begun to study homologs of SRP and the SRP receptor in E. coli. The reduced complexity of the SRP pathway as well as the ease of performing genetic experiments in this organism makes it is an attractive model system. The role of SRP homologs in protein export has been controversial in part because substantial evidence suggests that many proteins can be targeted to the secretory pathway in an SRP-independent fashion. We have now obtained strong genetic evidence that SRP targets proteins to the previously identified Sec Y transport channel. These studies also suggest that inner membrane proteins may be among the substrates which are targeted via the SRP pathway. In other studies we have obtained surprising evidence that the E. coli homolog of SRP RNA may function-not only in an RNP complex with Ffh, but also either in a complex with another protein or as a free RNA at another stage of the SRP pathway.